Journal: Frontiers in Immunology

Article Title: The Envelope-Based Fusion Antigen GP120C14K Forming Hexamer-Like Structures Triggers T Cell and Neutralizing Antibody Responses Against HIV-1

doi: 10.3389/fimmu.2019.02793

Figure Lengend Snippet: Reactivity of GP120C14K fusion protein to bNAbs. Recognition of GP120C14K, GP120C, and BG505 SOSIP proteins to the quaternary conformational HIV-1 bNAb PGT151 (A) , broadly neutralizing antibodies PGT121 (B) , 257-D IV (C) and 10-1074 (D) and to the neutralizing monoclonal antibody C3 directed against the VACV 14K protein (E) .

Article Snippet: Briefly, 4 × 10 6 splenocytes or 10 6 cells from DLNs were stimulated with 5 μg/ml of clade C HIV-1 Env-1 peptide (a H-2 d -restricted CTL epitope with the sequence PADPNPQEM as previously described ( ) along with 1 μl/ml GolgiPlug (BD Biosciences), anti-CD107a-Alexa 488 (BD Biosciences) and monensin (1X; eBioscience), in RPMI 1640-10% FCS at 37°C for 6 h in a 96-well plate.

Techniques:

Journal: Frontiers in Immunology

Article Title: The Envelope-Based Fusion Antigen GP120C14K Forming Hexamer-Like Structures Triggers T Cell and Neutralizing Antibody Responses Against HIV-1

doi: 10.3389/fimmu.2019.02793

Figure Lengend Snippet: Generation and in vitro characterization of MVA-GP120C14K recombinant virus. (A) Scheme of the MVA-GP120C14K virus genome showing the insertion of the GP120C14K gene within the TK locus of the parental MVA genome. (B) PCR amplification of the viral DNA extracted from DF-1 cells infected with MVA-GP120C14K or MVA-wt viruses using the TK locus primers. The difference in size between the amplified segments from both viruses indicates the correct insertion of GP120C14K gene (2,215 bp) into the TK locus of MVA. The pCyA20-GP120C14K plasmid containing the fusion insert was used as a positive control template for the PCR reaction. (C) Growth kinetics of MVA-GP120C14K compared with that of MVA-GP120C and MVA-wt viruses in DF-1 cells (multiplicity of infection 0.01 PFU/cell). (D) Time-course expression of GP120C14K antigen by Western blot analysis. DF-1 cells were infected with MVA-GP120C14K virus at 5 PFU/cell and harvested at different times post-infection (6 and 24 hpi). Antigen expression was analyzed as reactivity against HIV-1 GP120 and VACV 14K proteins in the supernatant and cellular pellet (indicated as SN and P, respectively). The P2 stock of the virus was used as a positive control. (E) Sub-virion localization of GP120C and GP120C14K proteins by fractionation of the sucrose-purified MVA-GP120C and MVA-GP120C14K virus preparations, respectively. The unfractionated lysate virions (T.E.) and the various fractions (E1, E2, E3, and Core) obtained after sequential disruption by detergents were analyzed by Western-blot. Reactivity against HIV-1 GP120 and VACV 14K (bands corresponding to higher m.w being GP120C14K and lower m.w being 14K) are shown.

Article Snippet: Briefly, 4 × 10 6 splenocytes or 10 6 cells from DLNs were stimulated with 5 μg/ml of clade C HIV-1 Env-1 peptide (a H-2 d -restricted CTL epitope with the sequence PADPNPQEM as previously described ( ) along with 1 μl/ml GolgiPlug (BD Biosciences), anti-CD107a-Alexa 488 (BD Biosciences) and monensin (1X; eBioscience), in RPMI 1640-10% FCS at 37°C for 6 h in a 96-well plate.

Techniques: In Vitro, Recombinant, Amplification, Infection, Plasmid Preparation, Positive Control, Expressing, Western Blot, Fractionation, Purification

Journal: Frontiers in Immunology

Article Title: The Envelope-Based Fusion Antigen GP120C14K Forming Hexamer-Like Structures Triggers T Cell and Neutralizing Antibody Responses Against HIV-1

doi: 10.3389/fimmu.2019.02793

Figure Lengend Snippet: Time-course expression of HIV-1 Env SOSIP proteins expressed from MVA vectors by Western-blot analysis. (A) Monolayers of DF-1 cells were infected at 5 PFU/cell with MVA-WT, MVA-ZM197 SOSIP, or MVA-AMC011 SOSIP viruses. At the indicated times post-infection, infected cells were collected, cells extracts fractionated by 10% SDS-PAGE and analyzed by Western-blot using rabbit polyclonal anti-GP120 antibody to evaluate the expression of the SOSIP proteins in cellular pellet (P) and supernatant (SN). (B) bNAbs binding profile to Env antigens by flow cytometry. HeLa cells infected with MVA-GP120C14K, MVA-AMC011 SOSIP, MVA-ZM197 SOSIP, or MVA-GPN viruses were processed for flow cytometry as described under Materials and Methods using 10 μg/ml of the indicated primary human IgG anti-Env bNAb. Samples were acquired in a flow cytometer and geometric Mean Fluorescence Intensity (gMFI) values on the “live cells” gate were used to analyse the results.

Article Snippet: Briefly, 4 × 10 6 splenocytes or 10 6 cells from DLNs were stimulated with 5 μg/ml of clade C HIV-1 Env-1 peptide (a H-2 d -restricted CTL epitope with the sequence PADPNPQEM as previously described ( ) along with 1 μl/ml GolgiPlug (BD Biosciences), anti-CD107a-Alexa 488 (BD Biosciences) and monensin (1X; eBioscience), in RPMI 1640-10% FCS at 37°C for 6 h in a 96-well plate.

Techniques: Expressing, Western Blot, Infection, SDS Page, Binding Assay, Flow Cytometry, Fluorescence

Journal: Frontiers in Immunology

Article Title: The Envelope-Based Fusion Antigen GP120C14K Forming Hexamer-Like Structures Triggers T Cell and Neutralizing Antibody Responses Against HIV-1

doi: 10.3389/fimmu.2019.02793

Figure Lengend Snippet: HIV-1 Env-specific CD8 T cell immune responses elicited in spleen and DLNs after M+P or P+P prime/boost immunizations of mice with GP120C or GP120C14K. (A) Immunization schedule and immunization groups for the evaluation of the immunogenicity of GP120C14K over GP120C using a protein prime/protein boost (P/P) protocol or an MVA prime/protein boost (M/P) approach. “adj.” refers to the adjuvant mixture used. (B) HIV-1 Env-specific CD8 T cell responses measured at 10 days post-boost in the spleen (left) and DLNs (right) represented as the percentage of HIV-1-specific CD8 T cells secreting cytokines and/or expressing CD107a. * p < 0.05, *** p < 0.001. (C) Polyfunctional profile of the Env-specific CD8 T cell response at 10 days post-boost in the spleen. Data are represented with their respective confidence intervals for each of the population analyzed. The pie charts represent the relative proportions of cell populations that express four (burgundy), three (dark green), two (yellow), or one (gray) cytokine(s)/CD107a.

Article Snippet: Briefly, 4 × 10 6 splenocytes or 10 6 cells from DLNs were stimulated with 5 μg/ml of clade C HIV-1 Env-1 peptide (a H-2 d -restricted CTL epitope with the sequence PADPNPQEM as previously described ( ) along with 1 μl/ml GolgiPlug (BD Biosciences), anti-CD107a-Alexa 488 (BD Biosciences) and monensin (1X; eBioscience), in RPMI 1640-10% FCS at 37°C for 6 h in a 96-well plate.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: The Envelope-Based Fusion Antigen GP120C14K Forming Hexamer-Like Structures Triggers T Cell and Neutralizing Antibody Responses Against HIV-1

doi: 10.3389/fimmu.2019.02793

Figure Lengend Snippet: HIV-1 Env-specific GC B cell and Tfh immune responses elicited in DLNs and spleen, respectively, after M/P or P/P prime/boost immunizations of mice with GP120C or GP120C14K. Percentage of total (CD95+ IgG1+ GL7+) (A) or Env-specific (B) GC B cells in the DLNs of immunized animals. *** p < 0.001. Percentage of total (CXCR5+ PD1+) (C) or Env-specific (D) Tfh cells in the spleen of immunized animals. *** p < 0.001.

Article Snippet: Briefly, 4 × 10 6 splenocytes or 10 6 cells from DLNs were stimulated with 5 μg/ml of clade C HIV-1 Env-1 peptide (a H-2 d -restricted CTL epitope with the sequence PADPNPQEM as previously described ( ) along with 1 μl/ml GolgiPlug (BD Biosciences), anti-CD107a-Alexa 488 (BD Biosciences) and monensin (1X; eBioscience), in RPMI 1640-10% FCS at 37°C for 6 h in a 96-well plate.

Techniques:

Journal: Frontiers in Immunology

Article Title: The Envelope-Based Fusion Antigen GP120C14K Forming Hexamer-Like Structures Triggers T Cell and Neutralizing Antibody Responses Against HIV-1

doi: 10.3389/fimmu.2019.02793

Figure Lengend Snippet: Anti-Env total IgG antibody levels and HIV-1 neutralization capacity in immunized rabbits. (A) Immunization schedule and immunization groups ( n = 4) for the evaluation of the binding antibodies and the neutralization capacity of GP120C14K protein compared with the SOSIP constructs AMC011 and ZM197. (B) Anti-Env antibody levels (total IgG) in the serum of immunized rabbits at weeks 2, 6, 10, and 14 measured by ELISA. End-point titers of antibody responses obtained for each group were calculated from the reactivity shown against the corresponding protein and referred as the last dilution that provided an optical density value (at 450 nm) that is more than three times the value obtained at the same dilution by the naïve serum. (C) Neutralization titers (IC50) for the different immunization groups at weeks 0, 6, 10, and 14 against HIV-1 tier 1 (MW965.26 and SF162) and tier 2 (BG505 and sC22) viruses.

Article Snippet: Briefly, 4 × 10 6 splenocytes or 10 6 cells from DLNs were stimulated with 5 μg/ml of clade C HIV-1 Env-1 peptide (a H-2 d -restricted CTL epitope with the sequence PADPNPQEM as previously described ( ) along with 1 μl/ml GolgiPlug (BD Biosciences), anti-CD107a-Alexa 488 (BD Biosciences) and monensin (1X; eBioscience), in RPMI 1640-10% FCS at 37°C for 6 h in a 96-well plate.

Techniques: Neutralization, Binding Assay, Construct, Enzyme-linked Immunosorbent Assay